Various sample imaging methods are known from the art, for example optical microscopy (both optical reflection and transmission microscopy), fluorescence microscopy, and other methods. For example, the imaging can include scanning imaging or wide field imaging. Also, for example, imaging of samples can be applied to diagnose cancer. A known non-invasive method is to diagnose on samples having only a few cells by applying DNA-cytometry. An overview is provided by G. Haroske, F. Giroud, A. Reith and A. Bocking, in “1997 ESACP consensus report on diagnostic DNA image cytometry”, Analytical Cellular Pathology 17 (1998), 189-200. This method is based on the presence of numerical and/or structural chromosomal aberrations (aneuploidy), which are only detectable in tumor (neoplastic) tissue. Detection of DNA-aneuploidy allows the very early diagnosis of cancer, often years ahead of histological diagnoses on biopsies
A known DNA sample imaging method uses light of single wavelength. To that aim, the cell nucleus (DNA content) is coloured using a commonly known Feulgen staining method. The colouring step in this method, however, is time consuming, since it is based on a 4.5-hour Feulgen staining process. As a result, performing a brush biopsy and doing a real time measurement on the spot is not possible in this way.
As an alternative, it is known to use a fluorescent DNA staining, for example the marker DAPI that has a high specificity for binding to DNA.
The use of fluorescent DNA staining has certain advantages over Feulgen staining. For example, fluorescence allows for higher sensitivity of measurement than white light absorption. Moreover, most fluorescent dyes have a much faster staining time than the Feulgen staining process; for example, a DAPI-staining can be performed in several minutes.
However, an issue of using a fluorescent DNA dye is the fading of the fluorescence, due to irreversible photobleaching. It is known to use anti-fade agents to slow down the photobleaching to some extent, however, that only reduces the problem, but does not solve it.
Besides, in fluorescence DNA cytometry, focusing of a suitable illumination beam is done using the same light source and detection technique (fluorescence) as during the measurement itself. This will cause uncontrolled photobleaching of the fluorophores, and therefore leads to an increased Coefficient of Variance (CV) in the DNA cytometry measurement.